The physiological role of lipoxygenase in ethylene formation from 1-aminocyclopropane-1-carboxylic acid in oat leaves

In order to understand the physiological significance of the in-vitro lipoxygenase (EC 1.13.11.12)-mediated ethylene-forming system (J.F. Bousquet and K.V. Thimann 1984, Proc. Natl. Acad. Sci. USA 81, 1724–1727), its characteristics were compared to those of an in-vivo ethylene-forming system. While oat (Avena sativa L.) leaves, as other plant tissues, preferentially converted only one of the 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC) isomers to 1-butene, the lipoxygenase system converted all four AEC isomers to 1-butene with nearly equal efficiencies. While the in-vivo ethylene-forming system of oat leaves was saturable with ACC with a K_m of 16 M, the lipoxygenase system was not saturated with ACC even at 10 mM. In contrast to the in-vivo results, only 10% of the ACC consumed in the lipoxygenase system was converted to ethylene, indicating that the reaction is not specific for ethylene formation. Increased ACC-dependent ethylene production in oat leaves following pretreatment with linoleic acid has been inferred as evidence of the involvement of lipoxygenase in ethylene production. We found that pretreating oat leaves with linoleic acid resulted in increased ACC uptake and thereby increased ethylene production. A similar effect was observed with oleic acid, which is not a substrate of lipoxygenase. Since linoleic acid hydroperoxide can substitute for lipoxygenase and linoleic acid in this system, it is assumed that the alkoxy radicals generated during the decomposion of linoleic acid hydroperoxide are responsible for the degradation of ACC to ethylene. Our results collectively indicate that the reported lipoxygenase system is not the in-vivo ethylene-forming enzyme.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AEC 1-amino-2-ethylcyclopropane-1-carboxylic acid - Epps N-(2-hydroxyethyl)-piperazine-N-3-propanesulfonic acid - LH linoleic acid - LOOH linoleic acid hydroperoxide - pyridoxal-P pyridoxal-phosphate This work was presented at the 12th International Conference on Plant Growth Substances, Heidelberg, FRG, August 1985 (Abstract No. PO 5-52)     

Phenotypic character of the lipid hydroperoxide-resistant mutant: Positive relationship between flocculation and resistance against lipid hydroperoxide inSaccharomyces cerevisiae

A lipid hydroperoxide-resistant mutant was isolated from a strain ofSaccharomyces cerevisiae. The mutant was resistant to 1.5mm tert-butylhydroperoxide and 1.0mm linoleic acid hydroperoxide. It flocculated in a Ca²⁺-dependent manner and the resistance against lipid hydroperoxide was suppressed by mannose, which also inhibited flocculation. A positive relationship between the acquirement of, the flocculent phenotype and resistance against lipid hydroperoxide is suggested. A protein with a molecular weight of 33 kDa was found on the surface of the mutant cell.     

Arachidonic acid hydroperoxide stimulates lipid peroxidation in rat liver nuclei and chromatin fractions

Arachidonic acid, the most abundant polyunsaturated fatty acid in rat liver nuclei phospholipids is a major target of free radical attack, which induces lipid peroxidation. The non-enzymatic lipid peroxidation process in intact rat liver nuclei and in several chromatin fractions indicated that the most sensitive fatty acid for peroxidation is arachidonic acid C20:4 n-6. In this study, the effect of different amounts of arachidonic acid hydroperoxide on the lipid peroxidation of rat liver nuclei and chromatin fractions was studied; rat liver nuclei and chromatin fractions deprived of exogenous added hydroperoxide were utilized as control. The addition of arachidonic acid hydroperoxide to liver nuclei produces a marked increase in light emission that was hydroperoxide concentration dependent. The maximal peak of chemiluminescence displayed by the different chromatin fractions analyzed was observed between 20 and 80 min of incubation. The highest value of light emission was displayed by the high-density chromatin fractions, the 27.5 K fraction showed intermediate values of light emission, whereas the lowest density fraction produced very low chemiluminescence. A high correlation between arachidonic acid hydroperoxide concentration and chemiluminescence in the different chromatin fractions was observed. AC is Members of Carrera del Investigador Científico, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.     

Effects of acetyl-L-carnitine on the formation of fatty acid ethyl esters in brain and peripheral organs after short-term ethanol administration in rat

Increasing evidence suggests that Fatty acid ethyl esters (FAEE) play a central role in ethanol induced organ damage. In the current study we measured FAEE formation in rats after short-term oral administration of ethanol, in the presence and absence of pre-treatment with acetyl-L-carnitine. Ethanol treatment caused a significant increase in the levels of FAEE, particularly in the brain and heart, but also in the kidney and liver. Increases in FAEE were associated with a significant increase in FAEE synthase activity, GSH transferase activity, and lipid hydroperoxide levels. Pre-treatment with acetyl-L-carnitine resulted in a significant reduction of FAEE accumulation, decrease in FAEE synthase and GSH transferase activities, and lipid hydroperoxide levels. Administration of acetyl-L-carnitine greatly reduced the metabolic abnormalities due to non-oxidative ethanol metabolism, through an increment in lipid metabolism/turnover and by the modulation of the activities of enzymes associated with FAEE synthesis. These results suggest a potentially important pharmacological role for acetyl-L-carnitine in the prevention of alcohol-induced cellular damage.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Red cells exposed to t-butyl hydroperoxide undergo lipid peroxidation, haemoglobin degradation and hexose monophosphate-shunt stimulation. By using the lipid-soluble antioxidant 2,6-di-t-butyl-p-cresol, the relative contributions of t-butyl hydroperoxide and membrane lipid hydroperoxides to oxidative haemoglobin changes and hexose monophosphate-shunt stimulation were determined. About 90% of the haemoglobin changes and all of the hexose monophosphate-shunt stimulation were caused by t-butyl hydroperoxide. The remainder of the haemoglobin changes appeared to be due to reactions between haemoglobin and lipid hydroperoxides generated during membrane peroxidation. After exposure of red cells to t-butyl hydroperoxide, no lipid hydroperoxides were detected iodimetrically, whether or not glucose was present in the incubation. Concentrations of 2,6-di-t-butyl-p-cresol, which almost totally suppressed lipid peroxidation, significantly inhibited haemoglobin binding to the membrane but had no significant effect on hexose monophosphate shunt stimulation, suggesting that lipid hydroperoxides had been decomposed by a reaction with haem or haem-protein and not enzymically via glutathione peroxidase. The mechanisms of lipid peroxidation and haemoglobin oxidation and the protective role of glucose were also investigated. In time-course studies of red cells containing oxyhaemoglobin, methaemoglobin or carbonmono-oxyhaemoglobin incubated without glucose and exposed to t-butyl hydroperoxide, haemoglobin oxidation paralleled both lipid peroxidation and t-butyl hydroperoxide consumption. Lipid peroxidation ceased when all t-butyl hydroperoxide was consumed, indicating that it was not autocatalytic and was driven by initiation events followed by rapid propagation and termination of chain reactions and rapid non-enzymic decomposition of lipid hydroperoxides. Carbonmono-oxyhaemoglobin and oxyhaemoglobin were good promoters of peroxidation, whereas methaemoglobin relatively spared the membrane from peroxidation. The protective influence of glucose metabolism on the time course of t-butyl hydroperoxide-induced changes was greatest in carbonmono-oxyhaemoglobin-containing red cells followed in order by oxyhaemoglobin- and methaemoglobin-containing red cells. This is the reverse order of the reactivity of the hydroperoxide with haemoglobin, which is greatest with methaemoglobin. In studies exposing red cells to a wide range of t-butyl hydroperoxide concentrations, haemoglobin oxidation and lipid peroxidation did not occur until the cellular glutathione had been oxidized. The amount of lipid peroxidation per increment in added t-butyl hydroperoxide was greatest in red cells containing carbonmono-oxyhaemoglobin, followed in order by oxyhaemoglobin and methaemoglobin. Red cells containing oxyhaemoglobin and carbonmono-oxyhaemoglobin and exposed to increasing concentrations of t-butyl hydroperoxide became increasingly resistant to lipid peroxidation as methaemoglobin accumulated, supporting a relatively protective role for methaemoglobin. In the presence of glucose, higher levels of t-butyl hydroperoxide were required to induce lipid peroxidation and haemoglobin oxidation compared with incubations without glucose. Carbonmono-oxyhaemoglobin-containing red cells exposed to the highest levels of t-butyl hydroperoxide underwent haemolysis after a critical level of lipid peroxidation was reached. Inhibition of lipid peroxidation by 2,6-di-t-butyl-p-cresol below this critical level prevented haemolysis. Oxidative membrane damage appeared to be a more important determinant of haemolysis in vitro than haemoglobin degradation. The effects of various antioxidants and free-radical scavengers on lipid peroxidation in red cells or in ghosts plus methaemoglobin exposed to t-butyl hydroperoxide suggested that red-cell haemoglobin decomposed the hydroperoxide by a homolytic scission mechanism to t-butoxyl radicals.     

Impact of Surface Active Compounds on Iron Catalyzed Oxidation of Methyl Linolenate in AOT–Water–Hexadecane Systems

Edible oils contain minor surface active components that form micro-heterogeneous environments, such as reverse micelles, which can alter the rate and direction of chemical reactions. However, little is known about the role of these micro-heterogeneous environments on lipid oxidation of bulk oil. Our objective was to evaluate the ability of water, cumene hydroperoxide, oleic acid, and phosphatidylcholine to influence the structure of reverse micelles in a model oil system: sodium bis(2-ethylhexyl) sulfosuccinate (aerosol-OT; AOT) in n-hexadecane. The influence of reverse micelle structure on iron catalyzed lipid oxidation was determined using methyl linolenate as an oxidizable substrate. The size and shape of the reverse micelle were investigated by small-angle x-ray scattering, and water contents was determined by Karl Fischer titrations. Lipid hydroperoxides and thiobarbituric acid reactive substances were used to follow lipid oxidation. Our results showed that AOT formed spherical reverse micelles in hexadecane. The size of the reverse micelles increased with increased water or phosphatidylcholine concentration, but decreased upon addition of cumene hydroperoxide or oleic acid. Iron catalyzed oxidation of methyl linolenate in the reverse micelle system decreased with increasing water concentration. Addition of phosphatidylcholine into the reverse micelle systems decreased methyl linolenate oxidation compared to control and reverse micelles with added oleic acid. These results indicate that water, cumene hydroperoxide, oleic acid, and phosphatidylcholine can alter reverse micelle size and lipid oxidation rates. Understanding how these compounds influence reverse micelle structure and lipid oxidation rates could provide information on how to modify bulk oil systems to increase oxidative stability.     

Glucose promotes membrane cholesterol crystalline domain formation by lipid peroxidation

Oxidative damage to vascular cell membrane phospholipids causes physicochemical changes in membrane structure and lipid organization, contributing to atherogenesis. Oxidative stress combined with hyperglycemia has been shown to further increase the risk of vascular and metabolic diseases. In this study, the effects of glucose on oxidative stress-induced cholesterol domain formation were tested in model membranes containing polyunsaturated fatty acids and physiologic levels of cholesterol. Membrane structural changes, including cholesterol domain formation, were characterized by small angle X-ray scattering (SAXS) analysis and correlated with spectrophotometrically-determined lipid hydroperoxide levels. Glucose treatment resulted in a concentration-dependent increase in lipid hydroperoxide formation, which correlated with the formation of highly-ordered cholesterol crystalline domains (unit cell periodicity of 34 Å) as well as a decrease in overall membrane bilayer width. The effect of glucose on lipid peroxidation was further enhanced by increased levels of cholesterol. Treatment with free radical-scavenging agents inhibited the biochemical and structural effects of glucose, even at elevated cholesterol levels. These data demonstrate that glucose promotes changes in membrane organization, including cholesterol crystal formation, through lipid peroxidation.     

铁死亡机制及其在肿瘤治疗中的应用

铁死亡作为新发现的一种调节性细胞死亡,是一类铁依赖性脂质过氧化物累积所导致的细胞死亡方式。铁死亡与铁离子代谢、脂质代谢和氨基酸代谢存在密切关联。随着铁死亡相关分子机制的不断发展和完善,铁死亡在肿瘤治疗方面表现出良好的应用前景。本文将介绍铁死亡机制的研究进展及其在肿瘤治疗中的应用探索。     

In vivo formation of 1-malonylaminocyclopropane-1-carboxylic acid and its relationship to ethylene production in cocklebur seed segments: A tracer study with 1-amino-2-ethylcyclopropane-1-carboxylic acid

The in vivo formation of 1-malonylaminocyclopropane-1-carboxylic acid (malonyl-ACC) and its relationship to ethylene production in the axial tissue of cocklebur (Xanthium pennsylvanicum) seeds were investigated using the stereoisomers of the 2-ethyl derivative of ACC (AEC), as tracers of ACC. Of the four AEC isomers, the (1R, 2S)-isomer was converted most effectively to a malonyl conjugate as well as to 1-butene. Malonyl-AEC, once formed, was not decomposed, supporting the view that malonyl-ACC does not liberate free ACC for ethylene production in this tissue. d-Phenylalanine inhibited the formation of malonyl-AEC and, at the same time, promoted the evolution of 1-butene, whereas l-phenylalanine did not. Possibly, the d-amino-acid-stimulated ethylene production in cocklebur seed tissues is due to an increase in the amount of ACC available for ethylene production which results from the decrease of ACC malonylation in the tissues treated with d-amino acid. 2-Aminoisobutyric acid, a competitive inhibitor of ACC-ethylene conversion, did not affect the malonylation of AEC.     

Decreased lipid peroxidation in the rat kidney during gestation

Renal malonaldehyde content and lipid peroxidation, induced by ascorbate, NADPH and cumene hydroperoxide, are significantly decreased during gestation in rats. Lipid peroxidation tends to reach normal levels in the kidney post partum. In the renal mitochondria lipid peroxidation without co-factors and that induced by cumene hydroperoxide, ascorbate and NADPH is decreased during pregnancy. However, in the microsomes, only lipid peroxidation induced by NADPH and cumene hydroperoxide is affected. The observed decrease in lipid peroxidation during gestation is reflected by low levels of total lipid and phospholipid. Endogenous inhibitors of lipid peroxidation also increase during pregnancy.     

Lipid peroxidation in rat liver microsomes. II. Response of hydroperoxide formation to iron concentration

When rat liver microsomes were incubated with NADPH, the major products were hydroperoxides which increased with time indicating that endogenous iron content is able to promote lipid peroxidation. The addition of either 5 microM Fe2+ or Fe3+ ions strongly enhanced the hydroperoxide formation rate. However, due to the hydroperoxide breakdown, hydroperoxide concentration decreased with time in this case. Higher ferrous or ferric iron concentration did not change the situation much, in that both hydroperoxide breakdown and formation were similar to those when NADPH only was present in the incubation medium. After lipid peroxidation, analysis of fatty acids indicated that the highest amount of peroxidized PUFA occurred in the presence of 5 microM of either Fe2+ or Fe3+. This analysis also showed that after 8 min incubation with low iron concentration, PUFA depletion was about 77% of that observed after 20 min, whereas without any iron addition or in the presence of 30 microM of either Fe3+, PUFA decrease was only about 37% of that observed after 20 min. As far as the optimum Fe2+/Fe3+ ratio required to promote the initiation of microsomal lipid peroxidation in rat liver is concerned, the highest hydroperoxide formation was observed with a ratio ranging from 0.5 to 2. These results indicate that microsomal lipid peroxidation induced by endogenous iron is speeded up by the addition of low concentrations of either Fe2+ or Fe3+ ions, probably because free radicals generated by hydroperoxide breakdown catalyze the propagation process. In experimental conditions unfavourable to hydroperoxide breakdown the principal process is that of the initiation of lipid peroxidation.     

Ethylene evolution from tobacco leaves irradiated with UV-B

Seedlings of Nicotiana tabacum L. (cv. Petit Havana SR1) were grown in the presence or absence of ultraviolet-B (UV-B, 290–320 nm) irradiation. The evolution of ethylene from the leaves, the content of 1-aminocyclopropane-1-carboxylic acid (ACC), an endogenous precursor of ethylene, and the activity of ACC synthase, a rate-limiting step in the production of ethylene, were increased by UV-B irradiation. The time course of these increases was parallel with the emergence of damage that was estimated by measuring the chlorophyll (Chl) content and the leakage of ions from leaf cells. Treatment of leaves with aminoethoxy-vinyl-glycine (AVG), a specific inhibitor of ACC synthase, reduced the extent of damage caused by UV-B. These results suggest that ethylene acts on certain processes to cause damage in tobacco leaves irradiated with UV-B. Electronic Publication     

Regulation of 5-lipoxygenase enzyme activity

In this article, regulation of human 5-lipoxygenase enzyme activity is reviewed. First, structural properties and enzyme activities are described. This is followed by the activating factors: Ca2+, membranes, ATP, and lipid hydroperoxide. Also, studies on phosphorylation of 5-lipoxygenase and nuclear localization sequences are reviewed.     

Is peroxidase involved in ethylene biosynthesis?

Wheat (Triticum aestivum L. cv. Jubilar) coleoptile segments convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. This process is totally inhibited by nitrogen atmosphere and severely inhibited by free radical scavengers (sodium benzoate, ferulic acid), inhibitors of reactive -SH groups ( p -chlormercuribenzoate, iodoacetate), CoCl₂ and EDTA. Indole-3-acetic acid, aminoethoxyvinyl glycine, cycloheximide, actinomycin D, pyridoxal phosphate and NADH have no effect on ACC conversion to ethylene. Some in vivo characteristics of this conversion suggest that it could be catalyzed by peroxidase. However, isoperoxidase B1 isolated from wheat seedlings was not able to catalyze in vitro conversion of ACC to ethylene under a wide range of reaction conditions. Therefore, it is concluded that peroxidase is not directly involved in ethylene biosynthesis.     

Highly Sensitive Flow Injection Analysis of Lipid Hydroperoxides in Foodstuffs

Lipid hydroperoxides in oils and foods were measured by a flow injection analysis system with high sensitivity and selectivity. After sample injection, lipid hydroperoxides were reacted with diphenyl-1-pyrenylphosphine (DPPP) in a stainless steel coil, then the fluorescence intensity of DPPP oxide, that was produced by the reaction, was monitored. By this method, trilinolein hydroperoxide showed good linearity between 0.4 and 79pmol and their detection limits were 0.2pmol (signal-to-noise ratio = 3). The method made it possible to inject samples at 2-min intervals. There was a good agreement of the amounts of lipid hydroperoxides in oils and foods between by the batch method with DPPP and by the proposed method (coefficient of correlation: r = 0.999; n = 21; peroxide value = 0.09–167 meq/g). With this method, the calibration graph of trilinolein hydroperoxide was useful for all samples tested.     

Photooxidation of cell membranes in the presence of hematoporphyrin derivative: reactivity of phospholipid and cholesterol hydroperoxides with glutathione peroxidase

The susceptibility of photodynamically-generated lipid hydroperoxides to reductive inactivation by glutathione peroxidase (GPX) has been investigated, using hematoporphyrin derivative as a photosensitizing agent and the human erythrocyte ghost as a target membrane. Photoperoxidized ghosts were reactive in a glutathione peroxidase/reductase (GPX/GRD)-coupled assay only after phospholipid hydrolysis by phospholipase A2 (PLA2). However, enzymatically determined lipid hydroperoxide values were consistently approx. 40% lower than iodometrically determined values throughout the course of photooxidation. Moreover, when irradiated ghosts were analyzed iodometrically during PLA2/GSH/GPX treatment, a residual 30-40% of non-reactive lipid hydroperoxide was observed. The possibility that cholesterol product(s) account for the non-reactive lipid hydroperoxide was examined by tracking cholesterol hydroperoxides in [14C]cholesterol-labeled ghosts. The sum of cholesterol hydroperoxides and GPX/GRD-detectable lipid hydroperoxides was found to agree closely with iodometrically determined lipid hydroperoxide throughout the course of irradiation. Thin-layer chromatography of total lipid extracts indicated that cholesterol hydroperoxide was unaffected by PLA2/GSH/GPX treatment, whereas most of the phospholipid peroxides were completely hydrolyzed and the released fatty acid peroxides were reduced to alcohols. It appears, therefore, that the GPX-resistant lipid hydroperoxides in photooxidized ghosts were derived primarily from cholesterol. Ascorbate plus Fe3+ produced a burst of free-radical lipid peroxidation in photooxidized, PLA2-treated ghosts. As expected for fatty acid hydroperoxide inactivation, the lipid peroxidation was inhibited by GSH/GPX, but only partially so, suggesting that cholesterol hydroperoxide-derived radicals play a major role in the reaction.     

Molecular mechanism of metal-independent decomposition of lipid hydroperoxide 13-HPODE by halogenated quinoid carcinogens

Halogenated quinones are a class of carcinogenic intermediates and newly identified chlorination disinfection by-products in drinking water. 13-Hydroperoxy-9,11-octadecadienoic acid (13-HPODE) is the most extensively studied endogenous lipid hydroperoxide. Although it is well known that the decomposition of 13-HPODE can be catalyzed by transition metal ions, it is not clear whether halogenated quinones could enhance its decomposition independent of metal ions and, if so, what the unique characteristics and similarities are. Here we show that 2,5-dichloro-1,4-benzoquinone (DCBQ) could markedly enhance the decomposition of 13-HPODE and formation of reactive lipid alkyl radicals such as pentyl and 7-carboxyheptyl radicals, and the genotoxic 4-hydroxy-2-nonenal (HNE), through the complementary application of ESR spin trapping, HPLC–MS, and GC–MS methods. Interestingly, two chloroquinone–lipid alkoxyl conjugates were also detected and identified from the reaction between DCBQ and 13-HPODE. Analogous results were observed with other halogenated quinones. This represents the first report that halogenated quinoid carcinogens can enhance the decomposition of the endogenous lipid hydroperoxide 13-HPODE and formation of reactive lipid alkyl radicals and genotoxic HNE via a novel metal-independent nucleophilic substitution coupled with homolytic decomposition mechanism, which may partly explain their potential genotoxicity and carcinogenicity.     

Selective microdetermination of lipid hydroperoxides

A sensitive and selective assay for lipid hydroperoxides was developed based upon the activation by hydroperoxides of the cyclooxygenase activity of prostaglandin H synthase. The assay measures hydroperoxides directly by their stimulatory action on the cyclooxygenase and thus differs from the methods used currently which rely on the measurement of secondary products to estimate the amount of hydroperoxide. The present assay of enzymatic response was approximately linear in the range 10 to 150 pmol of added lipid hydroperoxide. This sensitivity for lipid peroxides is about 50-fold greater than that of the thiobarbiturate assay with fluorescence detection. When applied to samples of human plasma, the enzymatic assay indicated that the concentration of lipid hydroperoxides in normal subjects is 0.5 microM, more than 50-fold lower than estimated by the thiobarbiturate assay (30-50 microM). Nevertheless, the circulating concentration of 0.5 microM lipid hydroperoxide approaches that reported to have deleterious effects upon vascular prostacyclin synthase.     

Chemiluminescent assay of lipid hydroperoxides

The addition of luminol plus a catalyst such as peroxidase or a heme prosthetic group to a solution containing a small quantity of lipid hydroperoxides results in a flash of chemiluminescence, the intensity of which is a function of the hydroperoxide concentrations. Various protocols for lipid hydroperoxide assays have been described and we have studied conditions to increase their sensitivity and specificity. Plasma lipid hydroperoxide determinations require an extraction, since compounds present in plasma interfere with light emission. Moreover, the sensitivity of the assay is by the presence of hydrogen peroxide in the medium, which causes high background values. Catalase does not act on lipid hydroperoxides and can be used to eliminate hydrogen peroxide from the reaction medium. The determination requires a blank tube in which hydroperoxides are destroyed by incubating the sample with haematin plus ascorbate. The increase in the chemiluminescence of the assay tube caused by the presence of lipid hydroperoxides is then compared to the value obtained for an internal standard.     

Gluconeogenesis and the peroxisome

The interaction between hydroperoxides, cytochrome P450 and 8-anilino-1-naphthalenesulfonic acid (ANS) has been investigated. The addition of ANS to the cytochrome P450 solution did not effect the P450 Soret absorption peak or the reduced CO difference spectrum, suggesting that ANS may not bind to P450 heme directly. H2O2 or CuOOH alone did not effect ANS fluorescence and absorption spectra indicating that no detectable reaction occurs between hydroperoxide and ANS in the absence of P450. The reconstituted system of cytochrome P450, P450 reductase, lipid and NADPH did not mediate ANS metabolism. In the presence of P450, the addition of either H2O2 or CuOOH, however, leads to a decrease in ANS absorption around 258 nm and 350 nm indicating possible destruction of ANS. ANS destruction was confirmed with the disappearance of the ANS elution peak in the reverse phase HPLC profiles and with the changes in P450-bound ANS fluorescence intensity and the shift of max of ANS. Moreover , a very sensitive method to detect trace fluorescent products of ANS by thin layer chromatography has been developed based on the fact that ANS fluorescence is enhanced more than 1000-fold by the organic solvent butanol. A UV-sensitive fluorescent product was detected on thin layer chromatography profiles of the reaction mixtures. P450 was also observed to be modified by a fluorescent derivative of ANS, when the fluorescence was enhanced by butanol. These results also show that an organic compound which can not be metabolized by the reconstituted system of cytochrome P450 and NADPH-P450 reductase is metabolized by the reconstituted system of P450 and hydroperoxide, suggesting the activities of these two systems may not be completely comparable. (Mol Cell Biochem 167: 159-168, 1997)